Atrial Peptides

ABSTRACT

Novel atrial peptides having useful natriuretic activity are disclosed with the following amino acid sequence: 
     
         R.sub.1 
    
      -cys-phe-gly-gly-arg-ile-asp-arg-ile-gly-ala-gln-ser-gly-leu-gly-cys-asn-R 2   
     wherein 
     R 1  =H, ser, ser-ser, and 
     R 2  =OH, ser, ser-phe-arg, ser-phe-arg-tyr, 
     or the physiologically acceptable salts, esters or amides thereof.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation-in-part of copending application Ser. No.551,372, filed Nov. 10, 1983.

BACKGROUND OF THE INVENTION

This invention relates to novel atrial peptides having usefulnatriuretic activity.

It is known that the cells of the atrial myocardium in mammals containnumerous membrane-bound storage granules. These characteristic secretorygranules, which have been observed in the rat, dog, cat and human atria,resemble those which are in peptide-hormonal producing cells. See DeBoldet al., J. Histochem. Cytochem. 26, 1094-1102 (1978). It has beenreported that crude tissue extracts of atrial myocardium when injectedintravenously into non-diuretic rats produced a rapid and potentnatriuretic response. See DeBold et al., Life Sciences 28, 89-94 (1981).Partial purification of rat atrial homogenates with a brief boiling stepand fractionation on Sephadex® was achieved by Trippodo et al., Proc.Soc. Exp. Biol. Med. 170, 502-508 (1982). Natriuretic activity was foundby these investigators in the overall molecular weight range of 3600 to44,000 daltons and in peptide fractions of both the higher molecularweight range of 36,000-44,000 daltons and a lower molecular weight rangeof 3600-5500 daltons.

In a more recent publication, DeBold et al., Fed. Proc. 42(3), Abstract1870, page 611 (1983), report the purification of an atrial natriureticpeptide having a molecular weight of 5150 daltons and a sequence of 47amino acids which the investigators designated "Cardionatrin I". Threeadditional peaks with natriuretic activity were obtained by highperformance liquid chromatography (HPLC) procedures.

In a still later publication, Grammer et al., Biochem. Biophys Res.Commun. 116(2), 696-703, Oct. 31, 1983, disclose the partialpurification of a rat atrial natriuretic factor having a molecularweight of approximately 3800 and containing 36 amino acid residues.

Rat atrial extracts also have been fractionated into low molecularweight fractions (<10,000 daltons) and high molecular weight fractions(20,000-30,000 daltons) both of which in vitro relaxed smooth muscle andwere potent natriuretic agents when administered intravenously to rats.See Currie et al., Science 221, 71-73 (1983).

BRIEF DESCRIPTION OF THE INVENTION

In accordance with the present invention, novel peptides are providedwhich exhibit useful natriuretic activity. These biologically activepeptides have the following amino acid sequence:

    R.sub.1 -cys-phe-gly-gly-arg-ile-asp-arg-ile-gly-ala-gln-ser-gly-leu-gly-cys-asn-R.sub.2

wherein

R₁ =H, ser, ser-ser, and

R₂ =OH, ser, ser-phe-arg, ser-phe-arg-tyr,

or the physiologically acceptable salts, esters or amides thereof.

In the peptide structure, the amino acid components are designated byconventional abbreviations as follows:

    ______________________________________                                        Amino Acid    Abbreviated Designation                                         ______________________________________                                        L-Alanine     ala                                                             L-Arginine    arg                                                             L-Asparagine  asn                                                             L-Aspartic Acid                                                                             asp                                                             L-Cysteine    cys                                                             L-Glutamine   gln                                                             Glycine       gly                                                             L-Isoleucine  ile                                                             L-Leucine     leu                                                             L-Methionine  met                                                             L-Phenylalanine                                                                             phe                                                             L-Proline     pro                                                             L-Serine      ser                                                             L-Tyrosine    tyr                                                             ______________________________________                                    

The peptide materials of this invention have been isolated in a highlypurified form which did not exist in the rat myocardium from which theywere initially obtained. That is, they have been prepared in a formwhich is essentially free of other peptides, and free from othercellular components and tissue matter. These new atrial peptides exhibitphysiological characteristics which suggest that they are important tomedical science in the study of the endocrine system of the cardiacatria with respect to humoral agents for modulation of extracellularvolume, sodium and vascular resistance.

In particular, the novel peptides of this invention have indicatedtherapeutic use as a diuretic, natriuretic, renal vasodilator and smoothmuscle relaxant. That is, they exert profound effects on sodium, urinevolume, renal vasodilation and smooth muscle tone.

In brief, these novel peptides have been obtained by fractionation ofrat atrial extracts by gel filtration chromatography to provide a highand a low molecular weight fraction, both of which had usefulnatriuretic activity. The lower molecular weight peak was resolved byion-exchange chromatography into two peaks which possessed natriureticactivity and which either preferentially relaxed only the intestinal(chick rectum) muscle strips or which relaxed both vascular (rabbitaorta) and intestinal smooth muscle preparations. The intestinal smoothmuscle relaxant was separated into 4 peaks and purified to homogenity byreversed phase high performance liquid chromatography (HPLC). Sequenceanalysis established the structure of this serine-, glycine-rich peptideand demonstrated that the four biologically active peptides differedfrom each other by the lack of the first and the second amino terminalserine residues or of the C-terminal serine residue, respectively. The21 amino acid peptide was designated atriopeptin I, and the other threepeaks which relaxed intestinal strips and were natriuretic and diuretic,but which were ineffective on blood vessel strips, were designateddes-ser¹ -atriopeptin I, des-ser¹, ser² -atriopeptin I and des-ser²¹-atriopeptin I, respectively.

Similarly, the vascular smooth muscle relaxant which was the more potentnatriuretic-diuretic compound was resolved into two major peaks on HPLC.Surprisingly, the amino terminal 21 amino acids of both rabbit aortarelaxants was homogeneous with that of the intestinal relaxant but the23 amino acid peptide (designated atriopeptin II) possessed a phe-arg,and the 24 amino acid peptide (designated atriopeptin III) was extendedby a phe-arg-tyr at the carboxy terminus. This family of closely relatedpeptides is believed to be derived from a similar high molecular weightprecursor and the biological selectivity and potency of the smallerpeptides may be determined by the action of a limited sequentialproteolytic cleavage.

The shorter 21 amino acid peptide (designated atriopeptin I) relaxesintestinal but not vascular smooth muscle and is natriuretic anddiuretic in vivo. The second peptide (atriopeptin II) contained 23 aminoacids, i.e. the same 21 amino acids with a C-terminal phe-arg extensionwhich results in an agent that relaxes both vascular and intestinalsmooth muscle as well as being a potent natriuretic-diuretic in vivo.

DETAILED DESCRIPTION OF THE INVENTION

While the specification concludes with claims particularly pointing outand distinctly claiming the subject matter regarded as forming thepresent invention, it is believed that the invention will be betterunderstood from the following detailed description of preferredembodiments of the invention taken in connection with the accompanyingdrawings in which:

FIG. 1 is a graphical representation which shows the comparativeintestinal smooth muscle relaxant activity (chick rectum relaxation inmm) of the novel atrial pepetides in one embodiment of the invention.

FIG. 2 is a graphical representation which shows the comparativevascular smooth muscle relaxant activity (rabbit aorta relaxation in mm)of the novel atrial peptides in another embodiment of the invention.

The initial source material for isolating the peptides of this inventionwas the frozen rat heart. Over 2500 such rat hearts were subjected to asequence of steps for the isolation of the desired peptides. The stepsfor isolation can be briefly described as:

(a) Preparing a crude homogenate of mammalian atrial tissue andcentrifuging;

(b) Boiling the supernatant and centrifuging;

(c) Desalting the supernatant by gel filtration chromatography onSephadex® G-15 resin;

(d) Gel filtration chromatography of the protein fraction on Sephadex®G-75 resin;

(e) Ion exchange chromatography of the low molecular weight proteinfraction on SP-Sephadex® C-25 resin;

(f) High performance liquid chromatography (HPLC) of the two mainprotein fractions; and;

(g) Recovering the separated atrial peptide fractions.

The aforesaid Sephadex chromatography resins are well-known materialsavailable from Pharmacia Fine Chemicals, Piscataway, NJ.

Bioassays of the isolated peptides were made on rabbit aorta strips andon segments of chick rectum under physiologically acceptable conditions.Rabbit aorta strips maintained in tone by a continuous infusion ofnorepinephrine constituted a reliable and sensitive assay tissue. Use ofan isolated carbachol-(a muscarinic agent) contracted chick rectumpreparation, however, provided a more rapid and simpler assay thatfacilitated the testing of a larger number of samples.

Natriuretic activity of the isolated peptides was determined byinjecting intravenously in rats and determining the effect on fractionalsodium excretion in the urine.

These methods of determining biological activity (smooth muscle relaxantand natriuresis) as developed by a research group led by the inventorare further described by Currie et al., Science 221, 71-73 (1983).

The following detailed Examples will further illustrate the inventionalthough it will be understood that the invention is not limited tothese specific Examples. In the Examples, CRF means chick rectum factorwhile RAF means rabbit aorta factor.

EXAMPLE 1 Methods Relaxation of smooth muscle in vitro

Bioassay. Spiral strips of rabbit thoracic aorta and segments of chickrectum under 1 gm tension were continuously perfused at 10 ml/min withoxygenated Krebs-Henseleit solution (37°). Resting tone was induced byeither 2×10⁻⁸ M norepinephrine (aorta) or 2×10⁻⁸ M carbachol (rectum).The effects of test substances then were determined by application withmicropipets to the stream of medium flowing over the tissues, using asstandards nitroglycerin (aorta) and isoproterenol (rectum). Columnfractions were freeze dried and residues dissolved in phosphate bufferedsaline for bioassay.

Natriuresis. The natriuretic activity of extracts was determined in250-300 gm male Sprague-Dawley rats under dial-urethane anesthesia. Asuprapubic silastic bladder catheter was placed for urine collection anda tail vein catheter was used for infusion of 0.225% NaCl in 5% dextrosesolution at 38 μl/min. After an equilibration period of one hour, two 10minute (min) baseline urine collections were followed by rapidintravenous injection of the test substance and 3 more ten min urinecollections completed. Following a one hour re-equilibration period, asecond set of collections with a second injection of the test substanceswas completed. Urine volume was determined by weighing in taredcontainers. Sodium concentration was measured by flame photometry.

Preparation and purification of chick rectum factor (CRF) and rabbitaorta factor (RAF)

Homogenates were prepared from frozen atrial tissue in ˜30 g lotsderived from 200 rats by dispersion in 10 vol/wt tissue of phosphatebuffered saline in a 1 quart Waring blender (1 min) followed by PolytronPT20ST (20 seconds) at maximum speed. Suspensions were centrifuged 10min at 200×g. After heat treatment (10 ml aliquots in 18×150 mm testtubes immersed 10 min in a boiling water bath) this supernatant wascentrifuged again at 12000×g for 10 min. Acetic acid (glacial) then wasadded to the supernatant fluid to 0.5M, and the resultant suspensionclarified by a final centrifugation (27000×g for 15 min). Thesupernatant was chromatographed on a G-15 Sephadex column (8×36 cm) in0.5M acetic acid at 600 ml/hr and the protein fraction was concentratedby freeze drying. The combined material derived from 600 rats then wasdissolved in 0.5M acetic acid and supplied to a 5×90 cm G-75 Sephadexcolumn, eluting with 0.5M acetic acid at 96 ml/hr. Biological activity(smooth muscle relaxant and natriuresis) was found by assay methodologypreviously described by Currie et al., Science 221, 71-73 (1983) in twopeaks: a high (20,000 to 30,000) and a low (less than 10,000) molecularweight fraction.

Further purification of the low molecular weight fraction was achievedby ion exchange chromatography. The combined material from 1200 rats wasapplied to a column of SP-Sephadex C-25 (20 gm, dry weight, forming a5×7 cm column) in a 25 mM ammonium acetate/500 mM acetic acid. Thechromatogram was developed by application of a linear gradient ofammonium acetate increasing at 23.4 mM/hr at a flow rate of 96 ml/hr,with the acetic acid held at 500 mM. Biological activity was found onlyin two main fractions: one, designated peak CRF (eluted at 150 mMammonium acetate), which contained chick rectum relaxant factor (CRF),and the second, designated peak RAF (at 270 mM ammonium acetate), whichcontained rabbit aorta relaxant factor (RAF). Both fractions wereenriched in natriuretic activity as well.

The final stage of purification was accomplished by HPLC with UVmonitoring at 215 nm. The CRF and RAF fractions from the SP-Sephadexcolumn were lyophilized repeatedly to remove volatile materials,redissolved in 0.1% trifluoroacetic acid, and then HPLC was run on aBrownlee RP-300 Aquopore Column (4.6 mm×25 cm) using the followinggradients at 1.0 ml/min. CRF: 0→10%A over 3.8 min then 10%A→14.8%A over60 min then 14.8%A→16.4%A over 100 minutes. Three peaks of CRF activityeluted at 113.8 min. RAF: 0→16%A over 3.6 min then 16%A→22.4%A over 80minutes. A band of RAF activity eluted at 48.8 min. In all cases solventA=0.1% trifluoracetic acid/acetonitrile, B=0.1%, trifluoroacetic acid/H₂O. The bioactive fractions were reinjected on a Vydac column (C₁₈, 300 Åpore size, 4.6 mm×25 cm) acid eluted at 1.0 ml/min using a gradient of0→50%C over 50 min. The CRF sample resolved into one major peak (CRF I,29.3 min) and two minor peaks (CRF-II and -III, 29.5, 29.7 min). The RAFsample provided a major peak (RAF-I, 31.0 min) and a minor peak RAF-II,31.5 min). Products were lyophilized and when stored at -20°, exhibitedgood stability.

Edman degradation. The above-isolated polypeptides were sequentiallydegraded utilizing an Applied Biosystems Model 470A gas phase sequenceras described by Hunkapiller et al., Methods in Enzymol. 91 (1), Chapter36, Academic Press, N.Y., 1983. Several modifications included theomission of benzene as one of the solvents employed, and the replacementof methanol with acetonitrile as solvent 4 in the system. In addition,the conversion reagent utilized (reagent 4) was 25% trifluoracetic acid(in H₂ O v/v). Coupling times were reduced to about 600 sec total, whilecleavage times remained at 850 sec. Thirty or more cycles were completedin each run with one degradation each for CRF (665 pmoles output yield),reduced/alkylated CRF (600 pmoles), and RAF (1178 pmoles).Phenylthiohydantoin amino acids were identified using high performanceliquid chromatography as adapted from Hunkapiler and Hood, Methods inEnzymol. 91 (1), Chapter 43, Academic Press, N.Y., 1983. Averagerepetitive yield values of 91% were determined for those amino acidderivatives deemed worthy of accurate quantitation.

The method described, above, provides the sequence of steps followed inthe purification of 1,200 rat hearts. In order to assign a relativebiological activity, the relaxant activity of the atrial extracts wascompared to a nitroglycerin standard curve on the blood vessel (rabbitaorta) strips and to isoproterenol on the intestinal (chick rectum)strips. The initial crude homogenate of rat atria was too contaminatedto determine total activity. The 10 min. boiling step facilitated thepurification by eliminating a great deal of protein prior to desaltingon the Sephadex G-15 column. The low molecular weight fraction obtainedfrom the gel filtration column was further separated on the ion exchangecolumn based on differences in the preferential spasmolytic activity ofthe various fractions. Thus, testing a 10 μl aliquot of each fractiondemonstrated the presence of two peptides, one of which preferentiallyrelaxed the intestinal smooth muscle and one which at low dosepreferentially relaxed the blood vessel strip. However, a completedose-response analysis of both peaks indicated that chick rectumrelaxant exhibited a pronounced selectivity such that even high dose ofthis peptide was impotent as a blood vessel relaxant. On the other hand,the second peak produced concentration dependent relaxation of both theintestinal and vascular strips. The peak with the preferentialselectivity, i.e. the chick rectum relaxant, which also possessed thenatriuretic-diuretic activity in vivo, was further investigated asdescribed herein.

The lyophilized chick rectum active factor (CRF) obtained from the SPSephadex column was fractionated by reversed phase (Brownlee C₁₈) HPLC.The CRF was separated into three major fractions (I-III). Each fractionwas lyophilized and rechromatographed by HPLC on a VYDAC column (C₁₈,300 Å pore size). There was thus obtained 60 μg protein of CRF-I, 25 μgof CRF-II, and 25 μg of CRF-III. CRF-I was quantitated as a smoothmuscle relaxant and produced a concentration dependent relaxation butdid not relax the precontracted aorta strips. Intravenous administrationof CRF-1 protein produces an increase in urinary sodium concentration.

The purified CRF-I sample was analyzed by gas phase sequencing. Thesequences of the closely related low molecular weightspasmolytic/natriuretic peptides as determined in this Example 1 areshown in Table 1, below. The peptides exhibit a large number of serineand glycine residues. The CRF-II and CRF-III merely lack the aminoterminal one or two serines present in CRF-I, thereby suggesting thatthey are amino peptidase cleavage products. The intestinal receptorrecognition appears to be tolerant of losses on the amino termius sinceCRF-II and III are fully biologically active.

The purified low molecular weight peptide designated RAF-1 whichexhibited a preferential relaxation of vascular smooth muscle wasfurther analyzed with the gas phase sequentator. Surprisingly, thesequence of the initial 21 amino acids of RAF-I are exactly the same asthose observed for CRF-1. The major difference in the peptides residesin the carboxyl terminus. RAF-I is a potent vascular smooth musclerelaxant in vitro and a selective renal vasodilator in vivo. RAF-I alsoappears to be considerably more potent as a natriuretic substance thanCRF-I. The latter peptide requires large doses and produces a variablein vivo response.

                  TABLE 1                                                         ______________________________________                                        Amino Acid Sequences                                                          ______________________________________                                        CRF-I:                                                                        Ser--ser--cys--phe--gly--gly--arg--ile--asp--arg--                            ile--gly--ala--gln--ser--gly--leu--gly--cys--asn--                            ser                                                                           CRF-II: des--ser.sup.1 - CRF-I                                                CRF-III: des--ser.sup.1, ser.sup.2 - CRF-I                                    RAF-I:                                                                        Ser--ser--cys--phe--gly--gly--arg--ile--asp--arg--                            ile--gly--ala--gln--ser--gly--leu--gly--cys--asn--                            ser.sup.21 --phe--arg.sup.23                                                  ______________________________________                                    

RAF-I and CRF-I can readily be differentiated by charge (on ion exchangechromatography) and mobility on reversed phase HPLC. Thecarboxy-terminal sequence of the peptides dictates their biologicalspecificity. The shortened carboxy terminus on CRF-I restricts itsbiological activity to relaxation of the intestinal smooth muscle andweak natriuretic activity. This peptide does not relax isolated bloodvessel strips nor does it reduce renal resistance in vivo. On the otherhand, the extended carboxy terminal in RAF-I includes the structuralfeatures required for vascular receptor recognition and for theinitiation of natriuresis and diuresis. The homogeneous nature of aminoterminal 21 amino acids in CRF and RAF strongly indicate that they maybe derived from the same precursor peptide. Aminopeptidase cleavage ofat least the initial two serine residues does not radically compromisebiological activity. However, the site of attack on the carboxy portionof the atrial peptide appears to dictate the ultimate biologicalresponse. The proteolytic enzyme provides an ideal site for theregulation of the physiological actions of these spasmolytic(natriuretic) peptides.

EXAMPLE 2 Materials and Methods Purification Scheme

Fourteen hundred frozen rat atria (Biotrol, Indianapolis, IN) weretrimmed of extraneous tissue (153 gm wet wt), homogenized in 10 volumesof phosphate buffered saline in the presence of phenylmethylsulfonylfluoride (1 μ/ml, Sigma Chemical Company, St. Louis, MO) and centrifugedat 2500×g for 10 min. The supernatant was divided into 10 ml aliquotsand immersed in a 100° bath for 10 min and centrifuged at 10,000×g for10 min at 4°. The supernatant was adjusted to 0.5M acetic acid andapplied to a Sephadex G-15 column (8×36 cm) and eluted with 0.5M aceticacid (600 ml/hr). The column effluent was lyophilized and reconstitutedin 0.5M acetic acid, applied to a Sephadex G-75 column (5×90 cm) andeluted with 0.5M acetic acid at 96 ml/hr. The lyophilized low molecularweight fraction from the G-75 column was applied to SP-Sephadex C-25 (20g gel, 5×7 cm column) in 25 mM ammonium acetate/0.5M acetic acid andeluted with a linear gradient of ammonium acetate (23.4 mM/hr at 96ml/hr) in 0.5M acetic acid. Two biologically active fractions eluted at160 mM which relaxed intestinal but not vascular smooth muscle stripsand the other at 270 mM relaxed both vascular and intestinal assaystrips. Following lyophilization the low molecular weight peaks wereindividually purified by reversed phase high pressure liquidchromatography on a Brownlee RP-300 aquapore column (4.6 mm×25 cm) usinga mixture of solvent A (0.1% trifluoracetic acid/acetonitrile) and B(0.1% trifluoracetic acid/water) at 1.0 ml/min.

The fraction that eluted from the SP-sephadex column at 160 mM ammoniumacetate was run at 0 to 10% A over 3.8 min; then 10 to 14.8% A over 60min; then 14.8 to 16.4% A over 100 minutes. Atriopeptin I eluted at15.6% A, des-ser¹ -atriopeptin I eluted at 15.7% A, des-ser¹, ser²-atriopeptin I eluted at 15.7% A, and des-ser²¹ -atriopeptin I at 15.8%A. The SP-Sephadex fraction that eluted at 270 mM was separated on thesame gradient on the HPLC with atriopeptin II being recovered from agradient 0 to 16% A in 5.8 min and 16 to 22% in 80 min. at 19.6% A, andatriopeptin III at 21.1% A. The bioactive fractions were reapplied to aVydac octadecasilyl column (300 Å pore size, 4.6 mm×25 cm) and eluted at1.0 ml/min using a mixture of solvent A (0.05% trifluoroacetic acid inacetonitrile) and B (0.05% trifluoracetic acid in water) employing agradient 0 to 30% over 30 min. Atriopeptin I appeared at 29.5% A;des-ser¹ -atriopeptin I at 29.7% A; des-ser¹, ser² -atriopeptin I at29.7% A; des-ser²¹ -atriopeptin I at 29.9%A; atriopeptin II at 31.5% A;and atriopeptin III at 32% A from a gradient of 10 to 35% over 25 min.The polypeptides were sequentially degraded, utilizing an appliedBiosystem Model 470 A gas phase sequencer as described in Example 1.Thirty or more cycles were completed in each run with one degradationeach for: the reduced and alkylated atriopeptin I (600 pmoles outputyield); des-ser¹ -atriopeptin I (660 pmoles); des-ser²¹ -atriopeptin I(520 pmoles); des-ser¹, ser² -atriopeptin I (650 pmoles); atriopeptin II(1200 pmoles), and atriopeptin III (850 pmoles). The atriopeptins werereduced and alkylated by dissolving the peptide (0.5-5 nmoles) in 90 μlof 2% SDS (sodium dodecylsulfate) in 0.4M Tris acetate (pH 9.0); 10 μlof 100 mM dithiothreitol was added, flushed with N₂, capped andincubated at 37° for 60 min. Then 20 μl of a fresh solution of 120 mMiodoacetamide (which had been recrystallized 3×), flushed with N₂,capped and incubated at room temp. for 10 min, then transferred toboiled dialysis tubing and dialyzed against 0.1% SDS for 2 hrs andredialyzed overnight followed by lyophilization. Phenylthiohydantoinamino acids were identified using high performance liquid chromatographyas described in Example 1. Average repetitive cycle yields were greaterthan 90% for each cycle whose signal allowed accurate quantitation. Theprotein concentration of the purified peptides was determined by themethod of Lowry et al., J. Biol. Chem. 193, 265-276 (1951). The smoothmuscle bioassay technique was performed as described by Currie et al.,Science 221, 71-73 (1983). Briefly, spiral strips of rabbit thoracicaorta and chick rectum were continuously superperfused at 10 ml/min withoxygenated Krebs-Henseleit medium (37°). In order to detect relaxantsubstances a resting tone was induced by infusing the vascular smoothmuscle preparations with norepinephrine (2×10⁻⁸ M.)

The natriuretic-diuretic assay (U_(Na) V) percent of the baselinecontrol was performed. The natriuretic-diuretic assay (U_(Na) V-percentof baseline control) was performed in 250-300 gm Sprague-Dawley ratsanesthetized with 0.4 ml dial-urethane. A suprapubic silastic catheterwas placed in the bladder for urine collection and a tail vein catheterwas used for infusion of 0.225% NaCl in 5% dextrose at 38 μl/min. Afteran equilibration period of one hour, two 10 min baseline urinecollections were followed by rapid intravenous injection of the testsubstance and 3 more 10 min urine collections were completed. Urinevolume was determined by weighing in tared containers. Sodiumconcentration was measured by flame photometery.

RESULTS

The purification protocol employed to produce peptides pure enough forsequence analysis is shown in Table 2, below. The initial crudehomogenate of rat atria is too contaminated to determine totalbiological activity. The 10 min boiling step facilitated thepurification by eliminating a great deal of protein prior to desaltingon the Sephadex G-15 column. The low molecular weight fraction obtainedfrom the gel filtration column was further separated on the ion exchangecolumn based on differences in net charge and in the preferentialspasmolytic activity of the various fractions. Thus, the testing ofaliquots of each fraction demonstrated the presence of two major peptidefractions, one of which preferentially relaxed the intestinal smoothmuscle and one which at low doses relaxed both the blood vessel and theintestinal strip. The lyophilized chick rectum active factor obtainedfrom the SP-Sephadex column was fractionated by reversed phase (BrownleeC₁₈) HPLC into four fractions. Similarly, the peak that also possess thevascular relaxation activity was resolved into two main peaks(atriopeptin II and III). Each fraction was lyophilized andrechromatographed by HPLC on a VYDAC column and underwent sequenceanalysis. The sequences of the closely related low molecular weightspasmolytic/natriuretic peptides as determined in this Example 2 areshown in Table 3, below. The peptides exhibit a large number of serineand glycine residues and contain an internal disulfide ring. The fourpeptides that selectively act on intestinal but not vascular smoothmuscle differ from each other because of the lack at the amino terminalof one or two serine residues, or lack of a C-terminal serine. Thepeptides that are potent vascular smooth muscle relaxants as well asintestinal spasmolytics contain a carboxyl terminal extension of phe-argor a phe-arg-tyr in atriopeptin II and III, respectively.

A quantitative comparison of the biological activity of the variousatrial peptides indicates that intestinal receptor recognition istolerant of losses on the amino terminus since des-ser¹ -atriopeptin Iand des-ser¹, ser² -atriopeptin are active peptides. However, the lackof a phe-arg carboxy extension precludes vaso-relaxant activity andpartially reduces in vivo natriuretic-diuretic activity. The in vitroand in vivo potency of atriopeptin II and III are comparable, suggestingthat further extension of the C-terminus beyond arg may not furthersubstantially alter activity. FIG. 1 and 2 illustrate this quantitativecomparison of the biological activity of these atrial peptides by theassay methods utilizing the chick rectum and rabbit aorta, respectively,as described hereinbefore.

                  TABLE 2                                                         ______________________________________                                                        Specific    Total    (Re-                                              Total  Activity    Activity covery                                            Protein                                                                              u/mg        Units    %)                                       ______________________________________                                        Crude      5764 mg  7.7           44,000                                      Homogenate                                                                    Boiled Extract                                                                           728      154     (1X)  112,000                                                                              (100%)                               Sephadex G-15                                                                            262      265     (1.7X)                                                                              69,400 (62%)                                Sepahdex G-75                                                                            17.0     2,890   (18.8X)                                                                             49,100 (44%)                                SP-Sephadex                                                                   C-25:                                                                         Intestinal 1.54     11,300  (73.4X)                                                                             17,400 (16%)                                Relaxant                                                                      Vascular   1.20     7,620   (49.5X)                                                                             9,140  (8%)                                 Relaxant                                                                      HPLC Fractions                                                                AP-I       0.114    37,600  (244X)                                                                              4,280  (3.8%)                               Mixture*   0.049    107,000 (695X)                                                                              5,220  (4.6%)                               des--ser.sup.21 -AP I                                                                    0.073            62,500 (406X)                                                                       4,550  (4.1%)                               AP-II      0.081    52,400  (340X)                                                                              4,260  (3.8%)                               AP-III     0.061    62,500  (406X)                                                                              3,810  (3.3%)                               ______________________________________                                         Table 2. Purification of 153 g tissue (atria from 1400 rat hearts). The       biological activity was compared on intestinal smooth muscle (chick           rectum) strips. Quantitation achieved by performing dose response curves      with each peptide in comparison to the response to a standard curve           achieved with ispoproterenol (the intestinal relaxant). One unit of           activity was set to be equivalent to 1 ng of isoproterenol.                   *Mixture of des--ser.sup.1 -AP I and des--ser.sup.1, ser.sup.2 -AP I.         AP = Atriopeptin                                                              u = Units                                                                

                                      TABLE 3                                     __________________________________________________________________________     ##STR1##                                                                      ##STR2##                                                                      ##STR3##                                                                      ##STR4##                                                                      ##STR5##                                                                      ##STR6##                                                                     __________________________________________________________________________

The atrial peptides were tested in vivo in rats for natriuretic potencyfollowing intravenous injection of 2 μg of the six peptides.Atriopeptins II and III were equipotent and somewhat more active thanatriopeptin I. Further shortening of the 21 amino acid length of thepeptide by loss of serines at either the N- or C-terminus resulted in areduction of the nutriuretic-diuretic potency. The results of this invivo testing are set forth in Table 4, below.

                  TABLE 4                                                         ______________________________________                                                     Residue                                                          Peptide      No.       n      U.sub.Na V                                                                            p Value                                 ______________________________________                                        Atriopeptin (AP) I                                                                         1-21          7    1130 ± 38                                                                          <.05                                  des--ser.sup.1 -AP I                                                                       2-21                                                                                   *    6    275 ± 62                                                                           <.05                                  des--ser.sup.1, ser.sup.2 -AP I                                                            3-21                                                             des--ser.sup.21 -AP                                                                        1-20          8    235 ± 70                                                                           NS                                    Atriopeptin II                                                                             1-23          3    1869 ± 114                                                                          <.005                                Atriopeptin III                                                                            1-24          6    1241 ± 261                                                                         <.01                                  ______________________________________                                         Table 4. Comparative effectiveness of the atrial peptides as                  natriureticdiuretic substances in vivo. The U.sub.Na V data is presented      as % of the stable baseline values obtained immediately prior to injectio     of the peptide. The baseline U.sub.Na V was 0.21 ± 0.05μ                equivalents/min.                                                              *Mixture of des--ser.sup.1 -AP I and des--ser.sup.1, ser.sup.2 -AP I.         NS = not significant                                                     

In the isolated perfused rat kidney, atriopeptin II and atriopeptin IIIproduced a concentration dependent renal vasodilation. The peptideswhich lack the phe-arg C-terminal (i.e., the atriopeptin I family ofpeptides) were very much less active renal vasodilators.

Various other examples and modifications of the foregoing examples willbe apparent to the person skilled in the art after reading the presentdisclosure without departing from the spirit and scope of the invention,and it is intended that all such examples or modifications be includedwithin the scope of the appended claims. Thus, variations in length andcomposition of the end groups (R₁ or R₂) of the peptides or in theindividual amino acids of the peptides which do not adversely ordetrimentally affect their biologic activity as defined herein areincluded within the scope of the appended claims.

What is claimed is:
 1. A peptide having potent natriuretic activitycomprising the following amino acid sequence:

    R.sub.1 -cys-phe-gly-gly-arg-ile-asp-arg-ile-gly-ala-gln-ser-gly-leu-gly-cys-asn-R.sub.2

wherein R₁ =H, ser, ser-ser, and R₂ =OH, ser, ser-phe-arg,ser-phe-arg-tyr,or the physiologically acceptable salts, esters oramides thereof.
 2. The peptide of claim 1 wherein R₁ is ser-ser and R₂is ser and which also exhibits intestinal smooth muscle relaxantactivity.
 3. The peptide of claim 1 wherein R₁ is ser and R₂ is ser andwhich also exhibits intestinal smooth muscle relaxant activity.
 4. Thepeptide of claim 1 wherein R₁ is H and R₂ is ser and which also exhibitsintestinal smooth muscle relaxant activity.
 5. The peptide of claim 1wherein R₁ is ser-ser and R₂ is OH and which also exhibits intestinalsmooth muscle relaxant activity.
 6. The peptide of claim 1 wherein R₁ isser-ser and R₂ is ser-phe-arg and which also exhibits vascular smoothmuscle relaxant activity.
 7. The peptide of claim 1 wherein R₁ isser-ser and R₂ is ser-phe-arg-tyr and which also exhibits vascularsmooth muscle relaxant activity.
 8. A therapeutic composition of matterfor producing diuresis or natriuresis comprising a therapeuticallyeffective amount of the peptide of claim
 1. 9. A therapeutic compositionof matter for producing intestinal smooth muscle relaxant activitycomprising a therapeutically effective amount of the peptide of any ofclaims 2, 3, 4 or
 5. 10. A therapeutic composition of matter forproducing vascular smooth muscle relaxant activity comprising atherapeutically effective amount of the peptide of any of claims 6 or 7.11. A method for producing diuresis or natriuresis in a mammalcomprising administering to said mammal a therapeutically effectiveamount of a peptide as defined in claim
 1. 12. A method for producingintestinal smooth muscle relaxant activity in a mammal comprisingadministering to said mammal a therapeutically effective amount of apeptide as defined in any of claims 2, 3, 4 or
 5. 13. A method forproducing vascular smooth muscle relaxant activity in a mammalcomprising administering to said mammal a therapeutically effectiveamount of a peptide as defined in any of claims 6 or
 7. 14. Atherapeutic composition of matter for producing renal vasodilationcomprising a therapeutically effective amount of a peptide as defined inclaim 1 in which R₂ is ser-phe-arg or ser-phe-arg-tyr.
 15. A therapeuticcomposition of matter for producing renal vasodilation comprising atherapeutically effective amount of the peptide as defined in claim 14in which R₁ is ser-ser.
 16. A method for producing renal vasodilation ina mammal comprising administering to said mammal a therapeuticallyeffective amount of a peptide as defined in claim 1 in which R₂ isser-phe-arg or ser-phe-arg-tyr.
 17. A method for producing renalvasodilation in a mammal comprising administering to said mammal atherepeutically effective amount of a peptide as defined in claim 16 inwhich R₁ is ser-ser.